The role of NCOA4-mediated ferritinophagy in iron homeostasis and ferroptosis Iron is essential for nearly all forms of life given the requirement for iron in oxygen binding and transport, ATP production, and DNA replication and repair. Cells must maintain sufficient iron levels to support these processes; however, iron excess can be detrimental given the role of iron in generation of toxic reactive oxygen species. To achieve this balance, cellular iron is primarily stored in a non-toxic form in ferritin, a protein complex composed of 24 subunits of ferritin light and heavy chain subunits. However, the mechanism of iron release from ferritin in times of cellular demand was unclear until recently. We identified NCOA4 as the selective cargo receptor that mediates the autophagic degradation of ferritin (?ferritinophagy?) thereby releasing iron for use by the cell. Flux through the ferritinophagy pathway is dependent on NCOA4 protein levels which are regulated by intracellular iron levels. Under iron-replete cellular conditions, NCOA4 binding to HERC2, an E3 ubiquitin ligase, is increased, leading to proteasomal degradation of NCOA4. Decreased NCOA4 levels inhibit ferritinophagy thereby increasing ferritin iron storage. Under iron-deficient conditions, HERC2 binding to NCOA4 is decreased, leading to NCOA4 stabilization and induction of ferritinophagy for iron release. Ferritinophagy is involved in iron- dependent physiological processes such as erythropoiesis, where NCOA4 mediates ferritin iron release for mitochondrial heme synthesis. Recently, in cell culture models, ferritinophagy has been shown to regulate ferroptosis, a newly described form of iron-dependent cell death mediated by excess lipid peroxidation. Dysregulation of iron metabolism and ferroptosis have been described in hemochromatosis and acute ischemic kidney injury; however, little is known about the in vivo role of ferritinophagy in regulating sensitivity to ferroptosis. In the proposed work, we will examine the hypothesis that NCOA4 is a key regulator of cellular and systemic iron homeostasis with specific temporal and spatial roles in regulating iron homeostasis in physiology and pathophysiology. Specific Aim 1 will define the biochemical mechanisms regulating NCOA4 activity in cells using an integrated quantitative proteomic and cell biologic experimentation workflow. In specific aim 2, we will determine the temporal and spatial dependency of ferritinophagy in vivo for regulating systemic iron homeostasis and erythropoiesis using novel mouse models of NCOA4 knockout and over-expression. Finally, specific aim 3 will focus on examining a role for NCOA4 in regulating sensitivity to ferroptosis in vivo under pathophysiologic conditions. Together, the proposed in vitro and in vivo studies will identify fundamental roles of NCOA4 in sensing and responding to iron deficiency and overload and determine the utility of pursuing NCOA4 as a therapeutic target for multiple iron-related disorders.